牙髓干细胞研究进展

干细胞是一类具有自我更新、高度增生能力和多相分化潜能的细胞,能够产生高度分化的功能细胞,包括胚胎干细胞和成体干细胞。目前,已在体外成功地扩增出人体多种组织的成体干细胞,自从2000年Gronthos等[1]发现人牙髓组织中存在成体干细胞即牙髓干细胞（dental pulp stemcells,DPSCs）后,近十年来国内外学者对牙髓干细胞的培养、生物学特性、细胞表型、分化能力及重组为诱导性多潜能干细胞（induced pluripotent stem cells,iPSCs）等方面已进行了大量研究,本文就牙髓干细胞的研究现状综述如下。     

骨髓基质干细胞的单克隆化培养和多向分化的实验研究

目的通过对骨髓基质干细胞（marrow stromal cells，MSCs）的单克隆化培养、鉴定和扩增，获得从一个单细胞扩增出的大量均质MSCs，并研究其多向分化能力。方法无菌条件下获取大鼠骨髓细胞，在培养的过程中挑出相应的单细胞克隆。将挑出的单细胞克隆与饲养细胞混合培养，快速进行扩增和细胞表面标志的鉴定，并进一步行增殖和细胞分化实验。结果在含有特定饲养层细胞的培养条件下，单克隆来源的MSCs能在抑制分化的状态下大量扩增，并保持细胞的均质性；而且在这种培养条件下，MSCs的增殖能力明显增强，并能成功被诱导向软骨、神经样细胞分化。结论通过骨髓细胞的单克隆培养，获得了均质和多向分化潜能的MSCs，为进一步更精确的研究MSCs的生物学特性提供了基础。     

小鼠成体肝脏干细胞体外培养模型的建立

目的 观察正常成体小鼠肝细胞的增殖能力和分化潜能,分离成体小鼠肝脏内可能存在的干细胞或祖细胞,建立细胞培养模型.方法 应用改良的Seglen二步法灌注和离心分离肝脏细胞,用含血清的改良DMEM培养基进行培养,持续观察超过60 d.应用免疫荧光技术对肝细胞及其形成的克隆进行Albumin、AFP和CKl9染色.结果 部分肝脏细胞培养第2～3天后活化,迅速增殖并形成细胞克隆,培养30 d后克隆内出现类似成熟的肝细胞,细胞克隆持续扩增超过60d.该类细胞培养第1天强阳性表达肝细胞标记物Albumin,培养第5天细胞克隆开始表达肝脏干细胞标记物AFP,第55天表达胆管细胞标记物CKl9.结论 在成体小鼠未损伤肝脏内存在一种成体肝脏祖细胞(adult hepatic progenitor cells,AHPCs),该细胞体外培养具有较强的增殖能力,可分化为肝细胞和胆管细胞,并成功建立了AHPCs的体外培养模型.     

不同代次小鼠脂肪干细胞增殖能力及体外成脂能力的实验研究

目的观察不同代次小鼠脂肪干细胞(mASCs)增殖能力及体外成脂能力,为组织工程种子细胞的合理选用提供实验依据。方法采用胶原酶消化小鼠腹股沟皮下脂肪组织,贴壁培养,体外传代扩增。实验采用原代(P0)、第2代(P2)和第5代(P5)细胞。观察不同代次mASCs的细胞形态及增殖能力;经高密度接种培养4周及成脂诱导2周,通过油红染色、RT-PCR检测,探讨其诱导成脂分化潜能及自发成脂能力。结果小鼠脂肪干细胞经传代后,外观主要呈长梭形及不规则形,不同代次的mASCs均具有很强的体外增殖能力;P0细胞具有极强的体外自发成脂能力,P2细胞自发成脂能力明显降低,至P5时基本没有体外自发成脂能力;P0、P2、P5细胞具有相似的诱导成脂分化潜能。结论随着培养代次的增加,mASCs自发成脂能力降低而诱导成脂分化潜能基本不变,扩增至第5代时仍维持了较强的增殖能力及良好的干细胞特性。在一定范围内mASCs体外传代扩增能明显减少其脂肪前体细胞的含量,有助于间充质干细胞纯化。     

长期体外培养对人脐血间充质干细胞成骨分化潜能的影响

目的观察体外长期培养对人脐血间充质干细胞（Human umbilical cord blood derived mesenchymal stem cells,UCB-MSCs）成骨分化潜能的影响,探讨其作为组织工程骨种子细胞的可行性。方法流式细胞技术观察长期体外培养的UCB-MSCs细胞表面抗原表达的变化规律,并通过Alizarin Red染色及碱性磷酸酶活性、骨钙蛋白含量和钙离子含量的检测观察长期体外培养对UCB-MSCs成骨特性的影响。结果第10代之前的UCB-MSCs能够高表达间充质干细胞表面标志物,7代之前的细胞体外增殖能力不随传代次数而发生明显变化。Alizarin Red染色及碱性磷酸酶活性、骨钙蛋白含量和钙离子含量的检测显示8代之前的UCB-MSCs仍能保持较强的成骨分化能力。结论7代之前的UCB-MSCs能保持稳定的体外成骨分化特性,有望成为较理想的组织工程骨种子细胞。     

牛磺熊去氧胆酸诱导牙髓干细胞的成骨分化实验研究

目的:研究牛磺熊去氧胆酸(Tauroursodeoxycholic acid,TUDCA)对牙髓干细胞(Dental pulp stem cells,DPSCs)增殖及成骨向分化的作用。方法:用不同质量浓度(0、10、100、30OnM)的TUDCA培养DPSCs,利用甲基噻唑基四唑(Methylthiazolylterrazolium,MTT)法计数检测不同浓度TUDCA对细胞增殖的影响;7d后测定不同浓度TUDCA诱导后的碱性磷酸酶(Alkaline phosphatase,ALP)活性;倒置相差显微镜下观察诱导后的DPSCs形态变化;应用茜素红染色法检测矿化结节的形成。结果:MTT法及ALP活性测定实验表明,随着TUDCA浓度的增加,ALP活性增强,细胞增殖率升高。茜素红染色显示TUDCA诱导培养28d后出现矿化结节。结论:TUDCA可促进DPSC增殖和成骨分化。     

传代种植密度对人间充质干细胞增殖及成骨分化的影响

目的 :研究人间充质干细胞 (mesenchymalstemcells ,MSCs)传代培养时 ,细胞种植密度对MSCs增殖及成骨诱导分化影响。方法 :将第 2代MSCs以 8× 10 3 /cm2 、 3× 10 3/cm2 、 8× 10 2 /cm2 密度接种 ,分析其生长曲线 ,并扩增培养 18d ,记录细胞扩增数量 ,再分析扩增后细胞的生长曲线和成骨诱导能力。结果 :人骨髓MSCs阴性表达ALP、CD3 4;在 8× 10 3/cm2 种植密度下 ,细胞倍增时间 40h ,18d后细胞数量扩增 (5 1± 13 )倍 ;在 3× 10 3 /cm2 种植密度下 ,细胞扩增 (2 8± 6)倍 ;在 8× 10 2 /cm2 种植密度下 ,细胞总数仅增加 (5± 3 )倍。在低密度下获得的细胞增殖能力强于高密度组 ,两组细胞均具有成骨诱导能力。结论 :种植密度对MSCs增殖有明显影响 ,快速扩增MSCs作为骨组织工程种子细胞 ,宜选择 8× 10 3 /cm2 种植密度。     

不同接种浓度的骨髓基质细胞体外生物学活性的研究

目的研究不同初始接种浓度对Beagle犬骨髓基质细胞（Bone marrow stromal cells，BMSCs）体外成骨能力的影响。方法将体外培养扩增的第1代BMSCs制成不同浓度（5×10^7cells／mL、5×10^6cells／mL、5×10^5cells／mL）的细胞悬液，分别接种于60mm培养皿，观察矿化结节形成的时间；并分别与胶原膜BME-10X复合，比较2d、5d、7d的碱性磷酸酶（Alkaline phosphatase，ALP）活性。结果细胞初始接种密度越高，形成矿化结节的时间就越短；在细胞载体复合物中，同一浓度组的ALP活性随培养时间的延长而增加；在相同天数，5×10^7cells／mL组与5×10^6cells／mL组、5×10^5cells／mL组之间ALP含量有显著差异，而5×10^6cells／mL组与5×10^5cells／mL组之间则无显著差别。结论BMSCs体外的成骨能力与细胞的接种密度有关，初始接种密度越高，其体外的成骨能力就越强。     

细胞接种密度对间充质干细胞成骨能力的影响

目的　研究初始细胞接种密度对人骨髓间充质干细胞成骨能力的影响。方法　将经体外培养扩增后的人骨髓间充质干细胞制成不同浓度的细胞悬液 ,与载体 (人冻干松质骨 )结合后植入裸鼠皮下 ,在植入后 2、 4和 8周对植入物进行组织学观察和骨量的测定。结果　植入物的骨量受初始细胞接种密度影响 ,稳定成骨的最低接种低密度是 1× 10 5cells/ml,植入物的骨量 ,在一定范围内随接种密度的升高而增加 ,接种密度≥ 5× 10 6cells/ml后骨量不再有显著变化。结论　人的骨髓间充质干细胞体内成骨能力受初始细胞接种密度的影响。     

瘦素对人牙髓干细胞骨向分化作用的实验研究

目的：探讨瘦素（leptin）对人牙髓干细胞（Human dental pulp stem cells,hDPSCs）增殖和骨向分化的影响。方法：采用酶消化法体外培养人牙髓干细胞,分别加阶梯浓度leptin处理,通过四唑盐（MTT）比色试验和碱性磷酸酶（Alkalinephosphatase,ALP）活性测试来检测瘦素对牙髓干细胞体外增殖分化的影响;再将人牙髓干细胞与羟基磷灰石/磷酸三钙材料制作为复合体植入免疫缺陷裸鼠,12周后采用组织学和免疫组化方法检测体内生成物。结果：瘦素对人牙髓干细胞细胞增殖无明显促进作用,但leptin能促进人牙髓干细胞碱性磷酸酶的活性表达;体内试验证明leptin可提高牙髓干细胞向成成牙本质细胞分化及生成类牙本质的能力。结论：瘦素对人牙髓干细胞的增殖活性无明显作用,但人牙髓干细胞与羟基磷灰石磷酸三钙制作为复合体,可明显促进人牙髓干细胞骨向分化。     

Initial cell plating density affects properties of human primary synovial mesenchymal stem cells

Synovial mesenchymal stem cells (MSCs) appear to be an attractive cell source in cartilage and meniscus regeneration because of their high proliferative and chondrogenic potentials. Two methods are used to culture synovial nucleated cells in the preparation of primary synovial MSCs. In one method, the cells are plated at low density to make cell colonies. In the other method, the cells are plated at high density. We investigated the effects of initial cell density on proliferation, surface markers, and multipotentiality, including chondrogenesis in primary synovial MSCs. Human synovium was obtained from the knee joints of patients with osteoarthritis after total knee arthroplasty. Immediately after enzyme digestion, the synovial nucleated cells were plated in densities of 10³, 10⁴, or 10⁵ cells/60‐cm² dish and cultured for 14 days. Proliferation, surface markers, chondrogenesis, adipogenesis, and calcification were examined in three populations. The cell colonies were distinct in the 10³ cells/dish group, faint in the 10⁴ cells/dish group, and obscure in the 10⁵ cells/dish group. The total number of cells/dish was positively related to plating density, whereas the fold increase was negatively related to plating density (n = 13). Among 12 surface markers, a negative relation to plating density was distinct in CD105. The cartilage pellet weight was negatively related to the initial plating density. The oil red‐o positive area and alizarin red positive area were positively related to the initial plating density. The initial cell plating density affected the properties of primary synovial MSCs. Synovial nucleated cells proliferated better when plated at low density, and the synovial MSCs obtained by this method contained a high chondrogenic potential. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1358–1367, 2019.     

Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density

The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm(2). The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.     

In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).     

Neural progenitor cells derived from the adult rat subventricular zone: characterization and transplantation

In order to fully characterize and determine the therapeutic potential of adult neural progenitor cells (NPCs), it is important to be able to isolate and study NPCs from animals such as rats, in which there are existing models of brain injury and disease. The focus of this study was to characterize the cultivation, differentiation, and transplantation of adult rat NPCs isolated from the subventricular zone of the lateral ventricles. We examined strategies for cell purification using a Percoll density gradient, and cell expansion using a range of maintenance medium and plating densities. Purification by Percoll gradient enriched a population of cells expressing nestin and SOX2, but resulted in a significant reduction in neurosphere generation. Culturing adult rat NPCs in Neurobasal-A media and plating at 200,000 cell/ml resulted in a higher percentage of cells surviving to generate neurospheres compared to culture in DMEM/F12 or NS-A media. On induction of differentiation, adult rat NPCs were capable of generating neurons, astrocytes, and oligodendrocytes in vitro that survived for up to 8 weeks, demonstrating multipotentiality of these cells. In addition, a population of cells continued to proliferate during the initial phase of differentiation, suggesting the presence of two populations of NPCs during differentiation. Cultured adult rat NPCs also survived and differentiated into astrocytes 6 weeks after transplantation into the striatum of the normal adult rat brain. In conclusion, we have optimized techniques that allow for the routine isolation, culture, and transplantation of multipotent NPCs derived from the adult rat SVZ.     

辛伐他汀对人牙髓干细胞增殖和分化的影响

目的:研究辛伐他汀对人牙髓干细胞(dental pulpstem cells,DPSCs)增殖和成骨分化的影响。方法:将第3代人DPSCs在矿化培养液中诱导培养,同时加入不同浓度的辛伐他汀(1×10-5mol/L、1×10-6mol/L、1×10-7mol/L、1×10-8mol/L),噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖情况,碱性磷酸酶试剂盒检测碱性磷酸酶(alkaline phosphatase,ALP)活性,茜素红染色鉴定成骨分化。结果:各浓度辛伐他汀均抑制人DPSCs增值,辛伐他汀浓度为1×10-5mol/L时,抑制作用最明显。适宜浓度辛伐他汀(1×10-6mol/L、1×10-7mol/L、1×10-8mol/L)促进人DPSCs向成骨细胞分化,其中,1×10-7mol/L的辛伐他汀促进ALP活性的作用最明显。结论:辛伐他汀抑制人DPSCs的增值,适宜浓度的辛伐他汀可有效促进人DPSCs的成骨分化。     

Cajal样细胞在人类后腹腔镜下肾癌根治手术标本的分布特点

目的探讨Cajal样细胞在经后腹腔镜肾癌根治手术标本中的形态和分布特点。方法收集我科2008年1～8月行后腹腔镜肾癌根治手术切下的肾脏和输尿管上段标本23例,分别对肾盏、肾盂和输尿管上段取材、石蜡包埋、切片,常规HE染色及CD117免疫组织化学染色,以正常结肠肠管的Cajal细胞作为阳性对照组,光学显微镜下观察两者形态学的异同,以及Cajal样细胞在上尿路的分布密度特点。结果形态学上人类上尿路Cajal样细胞与结肠肌壁间的Cajal细胞相似,呈梭形,CD117染色阳性,不同之处在于结肠的Cajal细胞主要位于内环、外纵肌间的肌间神经丛周围,而人类上尿路Cajal样细胞散在分布于上尿路的固有层和肌层间。分布密度上,肾盏、肾盂和输尿管上段依次为15.4±5.4、22.6±6.6和19.9±5.8个/cm2,肾盏区Cajal样细胞的分布密度明显小于肾盂和输尿管上段（P=0.000,P=0.014）,而肾盂和输尿管上段Cajal样细胞的分布密度差异无显著性（P=0.129）。结论人类上尿路存在Cajal样细胞,但其分布范围与胃肠道Cajal细胞不同,在肾盏、肾盂、输尿管上段的分布密度也不相同,这种差异可能与其功能有关。     

Absence of Glutamine Supplementation Prevents Differentiation of Murine Calvarial Osteoblasts to a Mineralizing Phenotype

Osteoblasts in vitro differentiate from a proliferating to a mineralizing phenotype upon transfer to a medium rich in beta-glycerophosphate and ascorbic acid. The nutritional requirements of the cells at different stages of this differentiation process are not known. In other cell types, nutritional supplementation during surgery can improve the outcome in terms of speed of patient recovery and prognosis. There is therefore the potential for supplementation at the site of fracture repair or bone grafting with critical osteoblast nutritional factors to potentially accelerate healing. In this study we investigate which common cell nutrients are required for the proliferating and mineralizing stages of osteoblast differentiation. Medium containing 5.5 mM glucose was sufficient to achieve maximal proliferation of primary calvarial osteoblasts and human osteoblast cell lines, with some added benefit of additional glutamine supplementation. However, when cells were stimulated to mineralize, glucose was insufficient to support their energetic requirements. Only when cells were supplemented with glucose together with glutamine were high levels of osteocalcin expression observed together with mineralized nodules in culture, suggesting that this would be a useful combination to assess in cultures of primary human osteoblasts to determine whether it may have beneficial effects during fracture surgery, bone grafting, and fixation of uncemented arthroplasty implants.     

HDAC抑制剂SAHA对人肝癌细胞分化诱导作用的研究

目的 探讨组蛋白去乙酰化酶(histone deacetylases,HDAC)抑制剂SAHA(suberoylanilide hydroxamic acid)对人肝癌SMMC-7721细胞的分化诱导作用.方法 倒置显微镜观察SAHA对SMMC-7721细胞形态的影响;MTT比色法测定SAHA对SMMC-7721细胞增殖的抑制情况;免疫细胞化学检测SAHA对SMMC-7721细胞中甲胎蛋白(AFP)和增殖细胞核抗原(PCNA)表达的影响;流式细胞术(FCM)分析细胞周期;RT-PCR方法榆测处理前后SMMC-7721细胞p21WAF1基因mRNA的表达变化.结果 实验组细胞增殖速度显著减慢,与正常细胞形念变化相似;MTT比色法测定结果显示不同浓度SAHA对SMMC-7721细胞的增殖均有抑制作用,并有明显的剂量依赖和时间依赖关系;免疫细胞化学检测显示SAHA能显著降低PCNA和AFP在SMMC-7721细胞中的表达;流式细胞仪检测结果显示,SMMC-7721细胞经SAHA处理后,G0/G1期细胞明显增加,S期细胞则明显减少,细胞被阻滞于G0/G1期;RT-PCR检测结果表明,实验组细胞中p21WAF1 mRNA的表达明显增加.结论 SAHA对人肝癌细胞具有显著的诱导分化作用,诱导肝癌细胞分化的机理可能与抑制HDAC的活性,上调p21 WAF1 mRNA表达,及阻滞肝癌细胞G0/G1期有关.     

Effects of transforming growth factor type β on expression of cytoskeletal proteins in endosteal mouse osteoblastic cells

Transforming growth factor β (TGFβ) has been shown to influence the growth and differentiation of many cell types in vitro. We have examined the effects of TGFβ on cell morphology and cytoskeletal organization in relation to parameters of cell proliferation and differentiation in endosteal osteoblastic cells isolated from mouse caudal vertebrae. Treatment of mouse osteoblastic cells cultured in serum free medium for 24 hours with TGFβ (1.5–30 ng/mL) slightly (− 23%) inhibited alkaline phosphatase activity. In parallel, TGFβ (0.5–30 ng/mL, 24 hours) greatly increased cell replication as evaluated by [³H]-thymidine incorporation into DNA (157% to 325% of controls). At a median dose (1.5 ng/mL) that affected both alkaline phosphatase and DNA synthesis (235% of controls) TGFβ induced rapid (six hours) cell respreading of quiescent mouse osteoblastic cells. This effect was associated with increased polymerization of actin, actinin, and tubulins, as evaluated by both biochemical and immunofluorescence methods. In addition, TGFβ (1.5 ng/mL) increased the de novo biosynthesis of actin, actinin, vimentin, and tubulins, as determined by [³⁵S] methionine labeling and fractionation of cytoskeletal proteins using two-dimensional gel electrophoresis. These effects were rapid and transient, as they occurred at six hours and were reversed after 24 hours of TGFβ exposure. The results indicate that the stimulatory effect of TGFβ on DNA synthesis in endosteal mouse osteoblastic cells is associated with a transient increase in cell spreading associated with enhanced polymerization and synthesis of cytoskeletal proteins.